Hepatitis C virus (HCV), the major etiologic agent of transfusion acquired non-A, non-B hepatitis, is responsible for approximately 150,000 new cases of acute viral hepatitis annually in the United States. Choo, et al., Science, 1989, 244, 359–362. There is a prevalence of 0.6 to 2.0% in western countries and up to 15% in some underdeveloped regions of the world. Heintges, et al., Hepatology, 1997, 26, 521–526. Approximately half of these infections progress to a chronic infection that can be associated with cirrhosis and/or hepatocellular carcinoma (Alter, et al., Science, 1992, 258, 135–140; and Alter, et al., New Eng. J. Med., 1992, 327, 1899–1905). In addition, HCV infection is an independent risk factor for the development of hepatocellular carcinoma as shown by the prevalence of anti-HCV antibodies (Colombo, et al., Lancet, 1989, ii, 1006–1008; Saito, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 6547–6549, Simonetti, et al., An. Int. Med., 1992, 116, 97–102; and Tsukuma, et al., New Eng. J. Med., 1993, 328, 1797–1801).
HCV is an enveloped, positive stranded RNA virus, approximately 9,500 nucleotides in length, which has recently been classified as a separate genus within the Flavivirus family. Heinz, Arch. Virol. (Suppl.), 1992, 4, 163–171. Different isolates show considerable nucleotide sequence diversity leading to the subdivision of HCV genomes into at least eight genotypes. Simmonds, et al., J. Gen. Virol., 1993, 74, 2391–2399. In all genotypes, the viral genome contains a large open reading frame (ORF) that encodes a precursor polyprotein of 3010 to 3033 amino acids of approximately 330 Kd. Choo, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 2451–2455; Inchauspe, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 10292–10296; Kato, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9524–9528; Okamoto. et al., J. Gen. Virol., 1991, 72, 2697–2704; and Takamizawa, et al., J. Gen. Virol., 1991, 65, 1105–1113.
Individual HCV polypeptides are produced by proteolytic processing of the precursor polypeptide to produce core (C), envelope (E1, E2) and non-structural (NS2–NS5) proteins. Bartenschlager, et al., J. Gen. Virol., 1993, 67, 3835–3844; Grakoui, et al., J. Gen. Virol., 1993, 67, 2832–2843; and Selby, et al., J. Gen. Virol., 1993, 74, 1103–1113. This proteolysis is catalyzed by a combination of both cellular and viral encoded proteases. The NS3 gene encodes for a serine protease which cleaves the viral polyprotein precursor post-transcriptionally at several functions and also serves as the viral helicase. The NS5 region encodes for the RNA-dependent RNA-Polymerase of the virus.
In addition to the translated region, the HCV genome also contains both a 5′ untranslated region (5′ UTR) and a 3′ untranslated region (3′ UTR). The 5′ UTR of 324 to 341 nucleotides represents the most highly conserved sequence among all HCV isolates reported to date. Han, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 1711–1715; and Bukh, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 4942–4946. This 5′ UTR has been postulated to contain important regulatory elements for replication and/or translation of HCV RNAs. The 5′ UTR also contains several small open reading frames (ORF) but there is presently no evidence to suggest that these ORF sequences are actually translated.
The cellular immune events involved in liver damage and viral clearance during HCV infection have only partially been defined. In an attempt to examine a potential pathogenic role of liver-infiltrating lymphocytes in patients with chronic HCV infection, Koziel, et al. examined the cytotoxic T lymphocyte (CTL) response of such cells and demonstrated an HLA class I-restricted CD8+ CTL response that was directed against both structural and non-structural regions of HCV polypeptides. Koziel, et al., J. Virol., 1993, 67, 7522–7532; and Koziel, et al., J. Immunol., 1992, 149, 3339–3344. Other investigators have also noted the existence of CTLs in peripheral blood mononuclear cell populations that recognize epitopes on core and the other viral related proteins during chronic HCV infection. Kita, et al., Hepatol., 1993, 18, 1039–1044; and Cerny, et al., Intl. Symp. Viral Hepatitis Liver Dis., 1993, 83 (abstr.). Botarelli, et al. (Botarelli, et al., Gastroenterol., 1993, 104, 580–587) and Ferrari, et al. (Ferrari, et al., Hepatol., 1994, 19, 286–295) found HLA class II-restricted CD4+ T cell-mediated proliferative responses to several recombinant proteins derived from different regions of HCV in patients with chronic HCV infection.
During active HCV infection, humoral and cellular immune responses have been shown to be polyclonal and multispecific and it is likely that the host immune response produced during persistent HCV infection is responsible, in part, for production of the liver cell injury. However, these immune responses may not be sufficiently broad based or vigorous enough to promote viral clearance and generate protective immunity in individuals with chronic HCV infection. Chisari, J. Clin. Invest., 1997, 99, 1472–1477. Those individuals who have recovered from acute HCV infection have recently been shown to develop strong proliferative CD4+ T cell responses directed against peptide derived from the nonstructural proteins. Missale, et al., J. Clin. Invest., 1996, 98, 706–714; and Diepolder, et al., Lancet, 1995, 346, 1006–1007. More important, the generation of HCV specific CTL activity appears to be associated with control of viral replication in individuals with chronic hepatitis. Rehermann, et al., J. Clin. Invest., 1996, 98, 1432–1440; and Nelson, et al., J. Immunol., 1997, 158, 1473–1481.
However, it is unknown if the nonstructural proteins NS3, NS4 and NS5 are sufficiently immunogenic to generate broad based and vigorous CTL-responses in vivo.
Presently, there is no universal, highly effective therapy of chronic HCV infection. Development of a vaccine strategy for HCV is complicated not only by the significant heterogeneity among HCV isolates, but also by the mixture of heterogeneous genomes within an isolate. Martell, et al., J. Virol., 1992, 66, 3225. In addition, the virus contains a highly variable envelope region. Effective therapy has been limited only to interferons. Carithers, et al., Hepatology, 1997, 26, 83S–88S. Indeed, approximately 8–10% of individuals treated with such agents respond and irradicate HCV from the liver. However, recent studies have revealed that individuals who recover from acute HCV infection develop substantial CD4+ T-cell proliferative responses against the nonstructural proteins as compared to those individuals who acquire persistent HCV infection. Missale, et al., J. Clin. Invest., 1996, 98, 706–714; and Diepolder, et al., Lancet, 1995, 346, 006–1007.
Direct injection of DNA into animals is a promising method for delivering specific antigens for immunization. Barry, et al., Bio Techniques, 1994, 16, 616–619; Davis, et al., Hum. Mol. Genet., 1993, 11, 1847–1851; Tang, et al., Nature, 1992, 356, 152–154; Wang, et al., J. Virol., 1993, 67, 3338–3344; and Wolff, et al., Science, 1990, 247, 1465–1468. This approach has been successfully used to generate protective immunity against influenza virus in mice and chickens, against bovine herpes virus I in mice and cattle and against rabies virus in mice. Cox, et al., J. Virol., 1993, 67, 5664–5667; Fynan, et al., DNA and Cell Biol., 1993, 12, 785–789; Ulmer, et al., Science, 1993, 259, 1745–1749; and Xiang, et al., Virol., 1994, 199, 132–140. In most cases, strong, yet highly variable, antibody and cytotoxic T-cell responses were associated with control of infection. Indeed, the potential to generate long-lasting memory CTLs without using a liver vector makes this approach particularly attractive compared with those involving killed-virus vaccines and generating a CTL response that not only protects against acute infection but also may have benefits in eradicating persistent viral infection. Wolff, et al., Science, 1990, 247, 1465–1468; Wolff. et al., Hum. Mol. Genet., 1992, 1, 363–369; Manthorpe, et al., Human Gene Therapy, 1993, 4, 419–431; Ulmer, et al., Science, 1993, 259, 1745–1749; Yankauckas, et al., DNA and Cell Biol., 1993, 12, 777–783; Montgomery, et al., DNA and Cell Biol., 1993, 12, 777–783; Fynan, et al., DNA and Cell Biol., 1993, 12, 785–789; Wang, et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 4156–4160; Wang, et al., DNA and Cell Biol., 1993, 12, 799–805; Xiang, et al., Virol., 1994, 199, 132–140; Davis, et al., Hum. Mol. Genet., 1993, 11, 1847–1851; Donnelly, et al., Nat. Med., 1995, 1, 583–587; Boyer, et al., Nat. Med., 1997, 3, 526–532; Tascon, et al., Nat. Med., 1996, 2, 888–892; and Huygen, et al., Nat. Med., 1996, 2, 893–898. The advantage of this method compared to immunizations with soluble recombinant proteins or peptides is the ability to induce a strong inflammatory CD4+ T cell response as well as cytotoxic T cell activity, presumably due to the intracellular processing of viral proteins into peptides and subsequent loading on MHC class I molecules in transfected cells and yet to be defined interactions with antigen presenting cells. In contrast, immunization with soluble protein leads primarily to a humoral immune response due to precessing through the MHC class II pathway. Immunization with synthetic peptides has several disadvantages since only a limited number of epitopes are available for stimulation of the host immune response. In contrast, all naturally occurring B and T cell epitopes encoded for each protein by the DNA construct of interest are presumably preserved for recognition by T cell receptors and therefore will presumably generate very broad based humoral and cellular immune responses. McDonnell, et al., N. Engl. J. Med., 1996, 334, 42–45.
Vaccination and immunization generally refer to the introduction of a non-virulent agent against which an individual's immune system can initiate an immune response which will then be available to defend against challenge by a pathogen. The immune system identifies invading “foreign” compositions and agents primarily by identifying proteins and other large molecules which are not normally present in the individual. The foreign protein represents a target against which the immune response is made.
PCT Patent Application PCT/US90/01348 discloses sequence information of clones of the HCV genome, amino acid sequences of HCV viral proteins and methods of making and using such compositions including anti-HCV vaccines comprising HCV proteins and peptides derived therefrom.
U.S. Pat. Nos. 5,830,876, 5,593,972, 5,739,118 and PCT Patent Application Serial Number PCT/US94/00899 filed Jan. 26, 1994, the disclosures of which are incorporated herein by reference in their entirety, each contain descriptions of genetic immunization protocols. Vaccines against HCV are disclosed in each.
There remains a need for vaccines useful to protect individuals against hepatitis C virus infection. There remains a need for methods of protecting individuals against hepatitis C virus infection.